PhD course in Molecular Biotechnologies
XX cycle (2005-2007)
PhD Thesis
“Hyaluronan role in Xenopus
laevis visceral skeleton
morphogenesis”
Candidate:
Paola Casini
Tutor:
INDEX
Preface ……… pg I Abstract ………. pg II
INTRODUCTION ..……… pg. 1
1. The “hyaluronic acid” alias “hyaluronan”……… pg. 1 1.1. Chemical properties ……….. pg. 1 1.2. Hyaluronan biosynthesis ……….. pg. 2 1.3. Hyaluronan catabolism ………..…… pg. 4 1.4. Hyaluronan is one of the main extracellular
matrix components ………..…… pg. 4 1.5. Intracellular hyaluronan ………... pg. 6 1.6. Hyaluronan functions ……… pg. 6 2. Hyaluronan shynthases ………. pg. 9 3. The hyaladherins ……….. pg. 10 3.1. The hyaluronan-receptor CD44 ………... pg. 11 3.2. The Receptor for Hyaluronan-Mediated Motility (RHAMM) ..…. pg. 13 3.3. Hyaluronan-receptors loss of function studies ………. pg. 14
4. The hyaluronan binding proteoglycan versican ……… pg. 15
5. The amphibian Xenopus laevis as model system ………. pg. 17 6. The neural crest cells ………... pg. 19
6.1. Neural crest migration ……….. pg. 19
6.1.1. Initiation of migration ………. pg. 19 6.1.2. Dispersion ……….... pg. 20 6.1.3. Cessation of migration ... pg. 24
7. The extracellular matrix and neural crest cells migration .. pg. 24
8. Cranial neural crest cells differentiation ... pg. 28 8.1. Hyaluronan in neural crest cells differentiation ……….. pg. 30
AIM OF THE WORK ……… pg. 32
1. Embryos handling ……….. pg. 33
2. Embryos microinjection and manipulation ……… pg. 33 2.1. Morpholino antisense oligos ……… pg. 34 2.2. CAPPED mRNAs ………. pg. 34
2.2.1. Constructs used for overexpression analysis:
linearization and capped mRNA transcription ………. pg. 35
3. In situ hybridization ………. pg. 35 3.1. Purification of plasmidic DNA ………. pg. 35
3.1.1. Constructs used for gene expression analysis: linearization and RNA transcription to generate
antisense RNA probes ……… pg. 35 3.2. Synthesis of digoxigenin (DIG) labeled probes ………. pg. 36 3.3. Whole mount in situ hybridization ……….………. pg. 36 3.4. In situ hybridization on frozen tissue sections ………. pg. 36
4. bHABC staining ……… pg. 37
5. Tunel assay ………. pg. 37
6. Alcian Blue staining ………. pg. 37 7. RNA extraction and RT-PCR ……… pg. 38
8. Solutions ………. pg. 39
RESULTS ……….. pg. 41
1. Gene expression profile of XHas1 and XHas2 during
cranial neural crest cells development ………. pg. 41 2. Hyaluronan detection in post-migratory cranial neural
crest cells ……….. pg. 45 3. Gene expression profile of XCD44 during cranial neural
crest cells development ………. pg. 46 4. Comparative analysis of the gene expression profile of
XHas1, XHas2 and XCD44 during cranial neural crest cell
development ………. pg. 47
5. XHas1 and XHas2 gene loss of function by a morpholino
antisense oligos based approach ………. pg. 48
5.1. XHas1 and XHas2 down-regulation interferes with cranial neural
5.2. XHas1 and XHas2 down-regulation cause apoptotic death of
post-migrated cranial neural crest cells ……….. pg. 51 5.3. XHas1 and XHas2 down-regulation leads to altered visceral
skeleton formation ……….. pg. 53 5.4. XHas1 and XHas2 loss of function control experiments ... pg. 55 5.5. Analysis of XHas1 and XHas2 double knock-down embryos……. pg. 57
6. Analysis of the XHas2 gain of function ……….. pg. 58 7. Analysis of the XCD44 loss of function ……… pg. 59 8. Expression profile of XRHAMM during cranial neural crest
cells development ………. pg. 62 9. Expression profile of Xversican during cranial neural crest
cells development ………. pg. 63
DISCUSSION ... pg. 67
1. XHas1 and XHas2 synthesize hyaluronan during Xenopus
laevis cranial neural crest cells development ……… pg. 67
2. Hyaluronan is required for proper migration of cranial
neural crest cells ……….. pg. 68 3. Hyaluronan contributes to the survival of cranial neural
crest cells in post-migratory stages ………... pg. 70 4. Hyaluronan is required for proper visceral skeleton
development ………. pg. 71
5. The XHas2 gain of function does not alter cranial neural
crest cells migration ………. pg. 73
6. CD44 is required for proper migration of cranial neural
crest cells ……… pg. 73
7. XRHAMM is expressed by migrating Xenopus laevis cranial
neural crest cells ………. pg. 75
8. Xversican is expressed during Xenopus laevis cranial neural crest cells development ……… pg. 76
CONCLUSION AND FUTURE PERSPECTIVES ……….. pg. 78
ACKNOWLEDGEMENTS ……… pg. 96
PUBLISHED PAPERS:
1. Ori M, Nardini M, Casini P, Perris R, Nardi I. (2006). XHas2
activity is required during somitogenesis and precursor cell migration in Xenopus development. Development. 133, 631-40.
2. Casini P, Ori M, Avenoso A, D’Ascola A, Traina P, Mattina W, Perris R, Campo GM, Calabroni A, Nardi I, Campo S. (2008, submitted
to Int. J. Dev. Biol.). Identification and gene expression of versican during early development of Xenopus.
I
PREFACE
During the first year of my PhD programme I took part in a project dealing with the identification of XCD44 gene expression pattern and its functional role during Xenopus laevis muscle development. These results are reported in the last part of this thesis as publications in “Development” journal (Ori, Nardini, Casini et al., 2006).
This PhD thesis describes all the work I have done in the last two years. In this period my efforts were focused on the project “Hyaluronan role in Xenopus
laevis visceral skeleton morphogenesis” that is the subject of this manuscript.
Moreover, as complementary studies, I report in the thesis the expression patter of the Xversican and XRHAMM genes during Xenopus laevis development. The
Xversican cDNA has been cloned in collaboration with the group of Prof.
Calatroni, University of Messina and the data regarding the identification and the gene expression of this gene are reported in the manuscript attached at the end of this thesis (Casini et al., 2008) actually submitted to an international peer review journal (“International Journal Developmental Biology”).
II
ABSTRACT
Hyaluronan is a crucial glycosaminoglycan of vertebrate extracellular matrix. In dynamic cellular systems, such as embryonic development, tissue regeneration and tumorigenesis, hyaluronan has been shown to influence cell behaviour, including cell migration, proliferation and differentiation both by assembling the interstitial matrices and by directly influencing cell behaviour via interaction with signal transducing receptors such as CD44.
We are using Xenopus laevis to study the role of hyaluronan and CD44
in vivo during cell migration and differentiation processes. The
spatio-temporal gene expression profile of the three known vertebrate hyaluronan synthases (Has1, Has2 and Has3) shows a very close conservation of
Xenopus laevis Has genes with that of mammals. Recently, we
demonstrated a critical role of XHas2 and XCD44 during muscle formation and precursor muscle cell migration. To further dissect the role of these molecules on migration and differentiation processes, during my PhD programme I then focused my attention on cranial neural crest cells (NCCs) development, knocking-down the XHas1, XHas2 and XCD44 gene functions. I showed that the hyaluronan synthases and the hyaluronan receptor present a dynamic expression pattern during cranial NCCs development suggesting multiple roles in the various steps of cranial NCCs migration and differentiation. I demonstrated that XHas1 and XHas2, in concert with XCD44, are involved in the NCCs migration and that hyaluronan, but not XCD44, is required in post-migratory stages to support cells survival. In order to investigate possible action mechanisms underlying hyaluronan function, I started to explore the possible functional interactions of hyaluronan with alternative receptors, such as RHAMM, and hyaluronan binding proteins such as the protoglycan versican.
On the whole the presented data demonstrated an unsuspected critical role of hyaluronan in the visceral skeleton morphogenesis and in particular in NCCs migration and differentiation. Moreover I showed for the first time the gene expression pattern of Xversican and XRHAMM in Xenopus laevis opening new working hypothesis that will be further investigated in the near future.
